1. Field of the Invention
The present invention relates to cell culture, and more particularly to a process for cell culture which achieves cell growth at a high cell density in a suspended state and is suitable for large-scale processing as well as for a culturing apparatus for the process.
2. Related Art Statement
Culture of mammalian cells in a large scale is an important technique for means of producing physiologically active substances used for medical drugs, diagnostics, etc.
Mammalian cells are roughly classified into those that can be proliferated in a suspended state and those that can grow only in a state adhered to a solid surface. As suspension culture apparatuses for the former case, there are employed a spinner flask, a roller bottle, a mechanical stirring type culture tank, etc. In the culture using apparatuses described above, however, cells are cultured in a fixed amount of nutrient sources so that a cell density of about 1.times.10.sup.6 cells/ml is obtained in many cell systems. As a method for culturing cells to a higher cell density level, there is known a method which comprises aseptically separating and collecting the cultured cells from the medium by means of centrifugation, etc., and resuspending and culturing the cells in a fresh medium. That is, cell wastes exhausted from cells and accumulated in a medium are withdrawn and a fresh medium is supplemented to supply nutrient sources; by doing so, a high cell density can be obtained. However, repeated centrifugation or the like permits various bacteria to readily invade into the culture system and its operations become complicated. Accordingly, it is desirable to continuously supply the culture system with nutrient sources and remove cell wastes from the culture system.
In order to realize this continuous supply of nutrient sources and removal of cell wastes, it is necessary to isolate cells stably over a long period of time without any cell damage. In recent years, some methods for high cell density cell culture in a large scale have been proposed to continuously supply nutrient sources and remove cell wastes.
For example, there are provided a method for exchanging a medium through filtration by rotating a cylindrical filter having the surface parallel to coaxial rotation while preventing the filter from getting clogged, and an apparatus suitable for the method (U.S. Pat. No. 3,647,632). There are also provided a method which comprises providing a pipe with an opening toward the bottom of a culture tank for precipitating cells in the tank and supplementing a fresh medium while exhausting out of the culture tank the supernatant obtained as the result of precipitation of the cells, and an apparatus for the method (Japanese Patent Application KOKOKU No. 61-36915). Furthermore, there are known a method for culturing cells which comprises using a semi-permeable membrane having permeability specific to selected nutrients, cell wastes or gas, passing a cell-containing medium and a cell-free medium along both sides of the semi-permeable membrane described above parallel to the membrane and supplying or discharging nutrients, cell wastes or gas into or from the cell-containing medium, and an apparatus used therefor (Japanese Patent Application KOKOKU No. 59-175877 and Japanese Patent Application KOKOKU NO. 59-175878).
According to the method for cell isolation using a filter, however, the filter tends to get easily clogged when used over long periods of time and any device is thus required to prevent the clogging; but the problem has not yet been satisfactorily overcome on an industrial level. Furthermore, the method using a precipitation tube involves a problem of cell damage due to oxygen starvation since cells settle in the precipitation tube for a long time. Further in the method using a semi-permeable membrane having permeability specific to the selected nutrient, cell wastes or gas, molecules are selectively permeated so that a pore diameter of the membrane is very small and a diffusion rate of nutrient or cell wastes through the membrane is small. Accordingly, it is difficult to obtain sufficient diffusion amounts of these substances at high density culture in a large scale. The semi-permeable membrane described above is also poor in mechanical strength and is thus unsuitable for culture over long periods of time.